Baicalin inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via the AKT/HIF-1α/p27-associated pathway

Abstract

Baicalin, a flavonoid compound purified from the dry roots of Scutellaria baicalensis Georgi, has been shown to possess various pharmacological actions. Previous studies have revealed that baicalin inhibits the growth of cancer cells through the induction of apoptosis. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by enhanced pulmonary artery smooth muscle cell (PASMCs) proliferation and suppressed apoptosis. However, the potential mechanism of baicalin in the regulation of PASMC proliferation and the prevention of cardiovascular diseases remains unexplored. To test the effects of baicalin on hypoxia, we used rats treated with or without baicalin (100 mg·kg⁻¹ each rat) at the beginning of the third week after hypoxia. Hemodynamic and pulmonary pathomorphology data showed that right ventricular systolic pressures (RVSP), the weight of the right ventricle/left ventricle plus septum (RV/LV + S) ratio and the medial width of pulmonary arterioles were much higher in chronic hypoxia. However, baicalin treatment repressed the elevation of RVSP, RV/LV + S and attenuated the pulmonary vascular structure remodeling (PVSR) of pulmonary arterioles induced by chronic hypoxia. Additionally, baicalin (10 and 20 μmol·L⁻¹) treatment suppressed the proliferation of PASMCs and attenuated the expression of hypoxia-inducible factor-α (HIF-α) under hypoxia exposure. Meanwhile, baicalin reversed the hypoxia-induced reduction of p27 and increased AKT/protein kinase B phosphorylation p-AKT both in vivo and in vitro. These results suggested that baicalin could effectively attenuate PVSR and hypoxic pulmonary hypertension.

Figure 1.

Baicalin attenuates chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling and inhibits rat pulmonary artery smooth muscle cell (PASMC) proliferation under hypoxia exposure. (A) Changes in right ventricular systolic pressure (RVSP); (B) Changes in the right ventricle/left ventricle plus septum (RV/LV + S) ratio; (C) Hematoxylin and eosin staining of pulmonary arterioles (original magnification ×20); (D) The ratio of intimal-to-medial areas of the vessel; (E) Hypoxia led to a significant increase in cell viability compared with the normoxic condition, while baicalin inhibited the effect in a concentration-dependent manner; (F) Hypoxia exposure significantly increased the cell proliferation. However, the hypoxia-induced proliferation of PASMCs was obviously inhibited by various dosages of the baicalin treatment. “Nor” means normoxia; “H” means hypoxia; “B” means baicalin. (^# p < 0.001; ** p < 0.01). All values are denoted as the mean ± SEM from six separate experiments.

Figure 2.

Effects of baicalin on p27/p21 and HIF-1α expression in rat lungs and cultured rat PASMCs. (A,B) Representative western blotting analysis of p27 and HIFα protein levels in rat lungs; (C,D) Representative western blotting analysis of p27 and HIFα protein levels in cultured rat PASMCs; (E,F) Western blotting analysis of p21 protein levels in rat lungs and in cultured rat PASMCs. “Nor” means normoxia; “H” means hypoxia; “B” means baicalin. (^# p < 0.001; ** p < 0.01; * p < 0.05). All values are denoted as the mean ± SEM from at least three separate experiments.

Figure 3.

The effects of baicalin on p-AKT expression in rat lungs and cultured rat PASMCs. (A) Representative western blotting analysis of p-AKT protein levels in rat lungs; (B) Representative western blotting analysis of p-AKT protein levels in cultured rat PASMCs. “Nor” means normoxia; “H” means hypoxia; “B” means baicalin. (^# p < 0.001; ** p < 0.01). All values are denoted as the mean ± SEM from at least three separate experiments.

Figure 4.

The effects of baicalin on mRNA expression in rat lungs and cultured PASMCs. (A,B) Analysis of p27 and HIF-1α mRNA levels in rat lungs; (C,D) Analysis of p27 and HIF-1α relative mRNA levels in cultured rat PASMCs. “Nor” means normoxia; “H” means hypoxia; “B” means baicalin. (^# p < 0.001; ** p < 0.01; * p < 0.05). All values are denoted as the mean ± SEM from at least three separate experiments.